Pectin based nanoparticles

ABSTRACT

This invention provides (a) nanoparticle made of: sugar-beet pectin; and a bioactive compound (such as a nutraceutical or a drug) bound to sugar-beet pectin. The invention further provides foods, beverages, including clear ones, or pharmaceutical preparations, which are supplemented with the nanoparticles made of: sugar-beet pectin; and a hydrophobic bioactive compound bound to sugar-beet pectin. The process of making the nanoparticles of the invention, and methods for supplementing foods or beverages or pharmaceutical preparations with bioactive compounds via the nanoparticles of the invention are also provided.

RELATED APPLICATIONS

This application is a continuation of U.S. patent application Ser. No.15/102,959 filed on Jun. 9, 2016, which is a National Phase of PCTPatent Application No. PCT/IL2014/051082 having International filingdate of Dec. 11, 2014, which claims the benefit of priority of U.S.Provisional Patent Application No. 61/915,019 filed on Dec. 12, 2013.The contents of the above applications are all incorporated by referenceas if fully set forth herein in their entirety.

FIELD OF INVENTION

This invention is directed to, inter alia, (1) a nanoparticlecomprising: pectin; and a bioactive compound bound to, entrapped orencapsulated within the pectin, and (2) beverages or foods supplementedwith the nanoparticles.

BACKGROUND OF THE INVENTION

Preventive medicine consists of measures taken to reduce the risk ofdiseases, including avoiding smoking, performing physical exercises andfollowing prudent dietary recommendations.

Recently, there is an increasing awareness that food may be eitherharmful or beneficial to health. This is based in part on a growingscientific understanding of the disease-preventive properties of foods,and in particular certain food components, known as nutraceuticals, suchas vitamins, omega-3 fatty acids, certain minerals and certainphytochemicals. Nutraceuticals are health-promoting bioactives. Theyhave been associated with the prevention and/or treatment of disorderslike cardio-vascular disease, cancer, hypertension, diabetes,osteoporosis, arthritis etc.

One way to increase the consumption of health promoting bioactives is toenrich foods and beverages that people normally consume withnutraceuticals. However, enriching foods with nutraceuticals may posegreat challenges, especially when the nutraceuticals are poorlywater-soluble, and are easily degradable.

The solubilization of hydrophobic health-promoting bioactives in cleardrinks is highly sought by beverage producers to provide added value forthe consumer, but it still poses tough challenges, particularly in shelfstable drinks. Most food grade surfactants, which may be used for thetask are synthetic e.g. the Tween (polysorbate) series, and thuspreclude an “all-natural ingredients” labeling. Other ways to enrichbeverages with hydrophobic nutraceuticals, like gum Arabic, milkproteins, soybean proteins and Maillard reaction conjugates are eitherexpensive, not always available, or are using allergenic components.

Clear drinks, which are consumed in large quantities, pose aparticularly important challenge because of the difficulty ofincorporating oil-soluble materials in a clear and stable aqueoussystem. The ideal vehicle for the task should be nano-sized to maintaintransparency, preferably ≤100 nm, and comprised of only natural,generally regarded as safe and inexpensive food components, capable ofsolubilizing and protecting hydrophobic biologically active molecules inaqueous media while retaining sensory qualities, and promotingbioavailability of hydrophobic biologically active molecules. Very fewsolutions for these challenging requirements have been suggested andnone has all the desired attributes.

Commercial pectins are often derived from citrus and apple pulps. Theseare characterized by high viscosity of low solids aqueous solutions andform gels in the presence of high solids sucrose solutions and/orcalcium under mildly acidic conditions. Historically, commercial pectinproduction from sugar beet commenced in Europe during World War II butceased when citrus and apple pulps again became available. Generally,sugar beet pectins were regarded as inferior and their function wasenhanced by conversion to low ester pectinic acids which produceionically crosslinked gels in the presence of polyvalent cations.

Spent sugar beet pulp consists largely of structural polysaccharidecomplexes associated with the primary cell wall of parenchymatoustissue. Subsequent extraction of spent beet pulp under hydrolyticconditions generates solubilized forms of the non-cellulosic componentswhose yield and chemical classification vary depending on the severityof hydrolysis condition and the specific extractive method employed.

Nanoencapsulation is a rapidly developing technology which has greatpotential to overcome solubility limitations, protect sensitivecompounds from degradation during production and shelf-life, maskundesired off-flavors, and promote bioavailability of encapsulatednutraceuticals.

Vitamin D and omega 3 were chosen as model hydrophobic nutraceuticalcompounds. Vitamin D is a fat soluble vitamin that has great importancefor calcium and phosphorus homeostasis. VD is also associated withcardiovascular health, cancer prevention, insulin sensitivity,regulation of immune function and decreased risk of autoimmune diseases.VD₃ is synthesized in the skin upon exposure to ultraviolet type-Bradiation. There are scarce natural dietary sources for VD, includingcertain fish oils and egg yolk. About 1 billion people worldwide are VDdeficient or insufficient, mainly due to avoidance of sun exposure toprevent melanoma, the use of sunscreen which blocks VD synthesis and lowdietary intake. Besides its low solubility in water, vitamin D issensitive to low pH, oxidation and heat.

Omega 3 fatty acids show remarkable preventive-medicine activities: theyreduce the risk of cardiovascular diseases and the metabolic syndrome,they lower blood pressure, serum cholesterol and triglyceride levels andthey are considered to have antithrombotic, antiatherogenic andanti-inflammatory properties. However, omega 3 fatty acids and theirester forms have very low aqueous solubility, and very high sensitivityto oxidation, resulting in undesired odors and flavors.

SUMMARY OF THE INVENTION

In one embodiment, the present invention provides a nanoparticlecomprising: (a) sugar-beet pectin; and (b) a bioactive compound boundto, entrapped or encapsulated within sugar-beet pectin. The bioactivecompound, in some embodiments, is a compound having maximal aqueoussolubility below 1 g/l. The bioactive compound, in some embodiments, isan oil-soluble vitamin, a polyunsaturated fatty acid or its ester, anantioxidant, a phytochemical, an Omega-3 fatty acid, or its esters, orany combination thereof.

In another embodiment, the present invention further provides acomposition comprising an aqueous liquid, a bioactive compound, andsugar-beet pectin. In some embodiments, the aqueous liquid is atransparent aqueous liquid such as a clear beverage. In one embodiment,a composition comprising an aqueous liquid, a bioactive compound, andsugar-beet pectin is devoid of an additional emulsifier.

In another embodiment, the present invention further provides a methodfor supplementing a subject with a bioactive compound, comprising thestep of administering to the subject a composition comprising: anaqueous liquid, a bioactive compound, and sugar-beet pectin bound to thebioactive compound, thereby supplementing a subject with a bioactivecompound. In some embodiments, the subject is afflicted with a diseaserequiring essential fatty acids support such as: a cardiovasculardisease, a reproductive disease, an immune disease, a nervous systemdisease, or any combination thereof.

In another embodiment, the present invention further provides a processfor preparing a nanoparticle comprising: a sugar-beet pectin; and abioactive compound bound to, entrapped or encapsulated within sugar-beetpectin, comprising the steps of preparing a first solution, a secondsolution, a mix of the first solution and the second solution; whereinthe first solution is prepared according to the steps of: (a) dissolvingsugar-beet pectin in water at a concentration of 0.1 to 100 g/L; (b)stirring the solution obtained in (a) for 10 minutes to 24 hours at 4 to40° C.; and (c) filtering the solution obtained in (b) through a filterhaving a cutoff of 0.1 to 1 microns; wherein the second solution isprepared according to the step of: dissolving the bioactive compound ina water-miscible organic food grade solvent; wherein the mix is preparedaccording to the step of: combining the first solution and the secondsolution by slowly adding said second solution into the first solutionwhile intensive stirring is applied, thereby preparing a nanoparticledispersion comprising: sugar-beet pectin; and a bioactive compound boundto, entrapped or encapsulated within sugar-beet pectin.

The nanoparticles of the invention may be dried by any method known inthe art, with or without the use of drying aids, such as saccharides,and reconstituted in water or aqueous solution to form a clear stablesolution or dispersion.

BRIEF DESCRIPTION OF THE DRAWINGS

FIG. 1. Is a graph providing estimation of the critical micellizationconcentration (CMC) of sugar beet pectin (SBP) from the onset of rise inthe I3/I1vs fluorescence of pyrene (CMC-1 mg/ml).

FIG. 2. Is a graph presenting the results of a curcumin—SBP bindingassay (k_(b)=(6.74±0.5)·10⁵M⁻¹), measured by fluorescence quenching ofthe tyrosine (Tyr) residue in the protein fraction of SBP, usingexcitation wavelength of 280 nm, and emission at 350 nm. Fluorescencewas measured at a constant SBP concentration (0.25 mg/ml of 3 mM PBS,pH6.8).

FIG. 3. Is a graph depicting the SBP protective effect. Residualpercentage of curcumin (with respect to time 0) during simulated shelflife, without and with protection by SBP. Curcumin was determined byabsorbance at 415 nm (maximal absorbance of curcumin) as determined by aUV-Visible light spectrophotometer.

DETAILED DESCRIPTION OF THE INVENTION

In one embodiment, the present invention provides a nanoparticle (or aplurality of nanoparticles) comprising: (a) sugar-beet pectin; and (b) abioactive compound bound to sugar-beet pectin. In another embodiment,the present invention further provides that the nanoparticle of theinvention is a nanocapsule comprising: (a) a nanoshell comprisingsugar-beet pectin; and (b) a core comprising a bioactive compound boundto—entrapped in or encapsulated by the sugar-beet pectinnanoshell.

In another embodiment, the present invention further provides that thepolysaccharide complex isolated by extractive hydrolysis of spent sugarbeet pulp as described herein will be referred to as a pectin, or amodified pectin. In another embodiment, “pectin” or “sugar beet pectin”include pectin and a protein naturally covalently bound to the pectin.In another embodiment, “a protein naturally covalently bound to thepectin” is a protein bound covalently to the pectin within a wild-typesugar beet plant.

In another embodiment, pectin according to the invention comprisesprotein, preferably (naturally) covalently bound to the pectin, forminga natural pectin-protein copolymer. In another embodiment, the saidprotein provides the hydrophobic domain of the natural pectin-proteincopolymer, capable of binding the active hydrophobic compound, as can bereadily determined by one of skill in the art. In another embodiment,pectin according to the invention comprises 0.5-3.5 weight % protein.

In one embodiment, pectin is a low methoxyl sugar-beet pectin. Inanother embodiment, pectin is a high methoxyl sugar-beet pectin. Inanother embodiment, pectin is at least a 40% esterified pectin. Inanother embodiment, pectin is 40% to 75% esterified pectin. In anotherembodiment, pectin is 60% to 78% esterified pectin. In anotherembodiment, sugar-beet pectin is a highly (2-O- and/or 3-O-galacturonicacid backbone) acetylated pectin (such as from sugar beet as describedherein). In another embodiment, pectin contains at least 65% by weightof galacturonic acid units.

In another embodiment, the nanoshell consists of a pectin. In anotherembodiment, the nanoshell comprises an additional polymer. In anotherembodiment, the additional polymer is a biodegradable polyester polymer.In another embodiment, the additional polymer is Poly-e-caprolactone(PCL). In another embodiment, the additional polymer is poly(lactide)(PLA). In another embodiment, the additional polymer ispoly(lactide-co-glicolide) (PLGA). In another embodiment, the additionalpolymer is poly(methacrylic acid). In another embodiment, the additionalpolymer is poly(N-vinyl Pyrrolidone). In another embodiment, theadditional polymer is a synthetic polymer. In another embodiment, theadditional polymer is a natural polymer. In another embodiment, theadditional polymer is a polysaccharide. In another embodiment, theadditional polymer is chitosan. In another embodiment, the additionalpolymer is gelatin. In another embodiment, the additional polymer issodium alginate. In another embodiment, the additional polymer isalbumin. In another embodiment, the nanoshell comprises apolysaccharide. In another embodiment, the nanoshell comprises asaccharide.

In another embodiment, the present invention further provides that ananocapsule encapsulates an inner liquid core, a solid core, or a partlyliquid and partly solid core. In another embodiment, the presentinvention further provides that a nanocapsule or a nanoparticle of theinvention has a diameter of 10 nm-100 nm. In another embodiment, thepresent invention further provides that a nanocapsule or a nanoparticleof the invention has a diameter of 5 nm-80 nm. In another embodiment,the present invention further provides that a nanocapsule or ananoparticle of the invention has a diameter of 10 nm-70 nm. In anotherembodiment, the present invention further provides that a nanocapsule ora nanoparticle of the invention has a diameter of 20 nm-60 nm.

In another embodiment, the present invention further provides that acore is devoid of a low molecular weight surfactant or is substantiallyfree of such a surfactant. In another embodiment, the present inventionfurther provides that the active substance such as a sparingly watersoluble substance is carried throughout the system properly and isreleased at the proper time and location. In another embodiment, thenanocapsules or nanoparticles of the invention are uniformly dispersedin water as an emulsion or within an emulsion.

In another embodiment, the present invention further provides that inits free form, the bioactive compound has maximal aqueous solubilitybelow 5 g/l (water). In another embodiment, the present inventionfurther provides that in its free form, the bioactive compound hasmaximal aqueous solubility below 3 g/l (water). In another embodiment,the present invention further provides that in its free form, thebioactive compound has maximal aqueous solubility below 2 g/l (water).In another embodiment, the present invention further provides that inits free form, the bioactive compound has maximal aqueous solubilitybelow 1.5 g/l (water). In another embodiment, the present inventionfurther provides that in its free form, the bioactive compound hasmaximal aqueous solubility below 0.5 g/l (water). In another embodiment,the present invention further provides that in its free form, thebioactive compound has maximal aqueous solubility below 0.1 g/l (water).

In another embodiment, the present invention further provides that thebioactive compound is an amino-acid or a peptide. In another embodiment,the present invention further provides that the bioactive compound is anon-polar amino-acid or a peptide. In another embodiment, the presentinvention further provides that the bioactive compound is a vitamin. Inanother embodiment, the present invention further provides that thebioactive compound is an oil-soluble vitamin. In another embodiment, thepresent invention further provides that the bioactive compound is apolyunsaturated fatty acid. In another embodiment, the present inventionfurther provides that the bioactive compound is an antioxidant. Inanother embodiment, the present invention further provides that thebioactive compound is phytochemical. In another embodiment, the presentinvention further provides that the bioactive compound is an ester ofany aforementioned compound described herein. In another embodiment, thebioactive compound is a lipid. In another embodiment, the bioactivecompound is a phospholipid. In another embodiment, the bioactivecompound is a glycolipid. In another embodiment, the bioactive compoundis a nutraceutical. In another embodiment, the bioactive compound is adrug. In another embodiment, the bioactive compound is a combination ofcompounds.

In another embodiment, the bioactive compound is an Omega-3 fatty acid.In another embodiment, the bioactive compound is an Omega-9 fatty acid.In another embodiment, the bioactive compound is an essential fattyacid. In another embodiment, the bioactive compound is an oil, such as,but not limited to, flax seed oil, fish oil or algae oil. In anotherembodiment, the bioactive compound is Linoleic Acid (LA). In anotherembodiment, the bioactive compound is Linolenic Acid (LNA). In anotherembodiment, the bioactive compound comprises LA, LNA, or both. Inanother embodiment, the bioactive compound is a sterol. In anotherembodiment, the bioactive compound is a phytosterol. In anotherembodiment, the bioactive compound is a zoosterol. In anotherembodiment, the bioactive compound is vitamin D.

In another embodiment, Zidovudine (INN) or azidothymidine (AZT) (alsocalled ZDV) is excluded from the present invention. In anotherembodiment, Zidovudine (INN) or azidothymidine (AZT) is not thebioactive compound of the present invention. In another embodiment,Itraconazole is excluded from the present invention. In anotherembodiment, Itraconazole is not the bioactive compound of the presentinvention. In another embodiment, the present invention is an oralcomposition. In another embodiment, the present invention does notinclude topical composition. In another embodiment, the presentinvention excludes oil based composition. In another embodiment, thepresent invention excludes lipid based composition.

In another embodiment, a composition as described herein is devoid ofZidovudine (INN) or azidothymidine (AZT) (also called ZDV). In anotherembodiment, a composition as described herein is devoid of Itraconazole.In another embodiment, a composition as described herein is devoid of anoil based composition. In another embodiment, a composition as describedherein is devoid of a lipid based composition.

In another embodiment, the present invention comprises less than 10% w/wlipids or oils. In another embodiment, the present invention comprisesless than 5% w/w lipids or oils. In another embodiment, the presentinvention comprises less than 2.5% w/w lipids or oils. In anotherembodiment, the present invention comprises less than 1% w/w lipids oroils. In another embodiment, the present invention comprises less than0.5% w/w lipids or oils. In another embodiment, the present inventioncomprises less than 0.1% w/w lipids or oils. In another embodiment, thepresent invention comprises less than 0.05% w/w lipids or oils. Inanother embodiment, the present invention comprises less than 0.01% w/wlipids or oils.

In another embodiment, the bioactive compound is vitamin A. In anotherembodiment, the bioactive compound is vitamin E. In another embodiment,the bioactive compound is vitamin K. In another embodiment, thebioactive compound is docosahexaenoic acid (DHA) or an ester thereof. Inanother embodiment, the bioactive compound is alpha lipoic acid. Inanother embodiment, the bioactive compound is a carotenoid. In anotherembodiment, the bioactive compound is beta-Carotene. In anotherembodiment, the bioactive compound is lutein. In another embodiment, thebioactive compound is lycopene. In another embodiment, the bioactivecompound is coenzyme Q10.

In another embodiment, the diameter of the nanoparticle or thenanocapsule is 5 to 200 nm. In another embodiment, the diameter of thenanoparticle or the nanocapsule is 10 to 100 nm. In another embodiment,the diameter of the nanoparticle or the nanocapsule is 10 to 80 nm. Inanother embodiment, the diameter of the nanoparticle or the nanocapsuleis 10 to 50 nm. In another embodiment, the diameter of the nanoparticleor the nanocapsule is 30 to 100 nm. In another embodiment, the diameterof the nanoparticle or the nanocapsule is 60 to 100 nm.

In another embodiment, pectin or sugar-beet pectin is in a pure form. Inanother embodiment, pectin or sugar-beet pectin is at least 90% pectin.In another embodiment, pectin is in a pure form. In another embodiment,pectin or sugar-beet pectin is at least 95% pectin. In anotherembodiment, pectin is at least 97% pectin. In another embodiment, pectinor sugar-beet pectin is at least 99% pectin.

In another embodiment, pectin is sugar beet pectin. In anotherembodiment, pectin is extracted via thermomechanical manipulation of thespent pulp under mildly acidic conditions whereby the hemicellulosecomplex becomes solubilized. In another embodiment, beet pectin ishighly acetylated and contains a moderate degree of feruloyl estersubstitution. In another embodiment, beet pectin includes hydrophobicmoieties coupled to extensive methyl esterification of D-galacturonicacid.

In another embodiment, beet pectin is obtained from aqueous extracts ofspent sugar beet pulp. In another embodiment, pectin as described hereinis an emulsifier and/or emulsion stabilizer.

In another embodiment, beet pectin is obtained from pulp or otherparenchymal cell-containing plant material that are isolated essentiallysimultaneously without substantial degradation. In another embodiment,beet pectin is obtained through hydrolysis of spent sugar beet pulp (orother plant material containing parenchymal cells in high proportion)under conditions of moderate pH and high temperature and may includephysical shearing. In another embodiment, acidic extraction of sugarbeet pulp is accomplished at pH's below about 4.5. In anotherembodiment, acidic extraction of sugar beet pulp is accomplished at pH'sbelow about 4.0.

In another embodiment, acidic extraction of sugar beet pulp isaccomplished at a temperature greater than 120° C. In anotherembodiment, acidic extraction of sugar beet pulp is accomplished at atemperature between 140 to 180° C. Thus, a sufficient combination of pH,reaction time and reaction temperature which allows the liberation ofpectin and arabinogalactan from spent sugar beet pulp (or otherparenchymal cell containing plant material) without a substantialdegradation.

In another embodiment, the isolation of the pectin components of sugarbeet pulp or other parenchymal cell containing plant material may beaccomplished in strongly alkaline conditions. Thus, combinations of high(strongly basic) pH, relatively high temperature and relatively shortreaction times may be employed for such isolation, enrichment, and/orpurification.

In another embodiment, sugar beet pectin is co-isolated with cellulosiccomponents of the materials from which it is derived. In anotherembodiment, sugar beet pulp, citrus pulp or other parenchymal cellcontaining material may be treated in such a way as to co-isolate bothparenchymal cell cellulose (PCC), and pectin components of those plantmaterials. In another embodiment, the resulting, combined materials maybe useful for any of the methods, and in any of the materials discussedabove under appropriate circumstances. In another embodiment, pectinincludes blends, mixtures or co-isolates form a natural gum havingproperties not unlike naturally-occurring gums well known to persons ofordinary skill in the food science art. In another embodiment,compositions of the present invention, include from about 0.01 to about10.0 wt.-% of sugar beet pectin. In another embodiment, compositions ofthe present invention, include from about 0.01 to about 1.0 wt.-% ofsugar beet pectin.

In another embodiment, the present invention further provides acomposition comprising the nanocapsules or the nanoparticles in anaqueous solution. In another embodiment, the present invention furtherprovides that the composition comprising the nanocapsules or thenanoparticles is a nanoemulsion. In another embodiment, the presentinvention further provides that the aqueous solution is a transparentaqueous liquid. In another embodiment, the present invention furtherprovides that the aqueous solution is a beverage. In another embodiment,the present invention further provides that the aqueous solution isdevoid of an additional emulsifier.

In another embodiment, the aqueous solution is transparent. In anotherembodiment, the aqueous solution comprises at least 70% by weight water.In another embodiment, the aqueous solution comprises at least 75% byweight water. In another embodiment, the aqueous solution comprises atleast 85% by weight water. In another embodiment, the aqueous solutioncomprises at least 90% by weight water. In another embodiment, theaqueous solution comprises at least 95% by weight water. In anotherembodiment, the aqueous solution comprises at least 98% by weight water.

In another embodiment, the present invention further provides that thecomposition of the invention is stable for at least 10 to 120 seconds atpH=2 to 8 and at temperature of 65 to 80° C. In another embodiment, thepresent invention further provides that the composition of the inventionis stable for at least 10 to 90 seconds at pH=2 to 8 and at temperatureof 70 to 80° C. In another embodiment, the present invention furtherprovides that the composition of the invention is stable for up to 60seconds at pH=2 to 8 and at temperature of 70 to 75° C. In anotherembodiment, the present invention further provides that the compositionof the invention is stable for at least 60 seconds at pH=2.5 and at atemperature of 72° C.

In another embodiment, the present invention further provides that thecomposition of the invention is stable at pH=2 to 8 during cooling froma temperature of 90° C. down to 25° C. within 30 min.

In another embodiment, the present invention further provides that thecomposition of the invention is stable for at least 2 months underappropriate storage conditions. In another embodiment, the presentinvention further provides that the composition of the invention isstable for at least 6 months under appropriate storage conditions. Inanother embodiment, the present invention further provides that thecomposition of the invention is stable for at least 12 months underappropriate storage conditions. In another embodiment, the presentinvention further provides that the composition of the invention isstable for at least 90 hours at pH=2-8 and at temperature of 4 to 35° C.In another embodiment, the present invention further provides that thecomposition of the invention is stable for up to 40 hours at pH 2 to 8and at temperature of 20 to 30° C. In another embodiment, the presentinvention further provides that the composition of the invention isstable for at least 24 to 36 hours at pH=2.5 and at temperature of 23 to27° C. In another embodiment, the present invention further providesthat the composition of the invention is stable for at least 24 hours atpH=2.5 and a temperature of 25° C. In another embodiment, the presentinvention further provides that the composition of the invention isstable for up to 24 hours at pH=2.5 and a temperature of 25° C.

In another embodiment, the phrase: “pectin and bioactive compound” issynonymous with the phrase “a bioactive compound bound to pectin”.

In another embodiment, the present invention further provides that theprocess of making a composition of the invention comprises the steps of:(1) mixing pectin and a bioactive compound in an aqueous solution; (2)freeze-drying or spray drying the solution comprising pectin and abioactive compound, thus obtaining a freeze-dried composition of pectinand a bioactive compound; (2) re-suspending the freeze-dried compositionof pectin and a bioactive compound in an aqueous solution.

In another embodiment, the present invention further provides that thebioactive compound is present at a concentration of 0.01 microgram/ml to10 mg/ml. In another embodiment, the present invention further providesthat the bioactive compound is present at a concentration of 0.5 mg/mlto 5 mg/ml. In another embodiment, the present invention furtherprovides that the bioactive compound is present at a concentration of 1microgram/ml to 1 mg/ml. In another embodiment, the present inventionfurther provides that the bioactive compound is present at aconcentration of 100 microgram/ml to 1 mg/ml. In another embodiment, thepresent invention further provides that the bioactive compound ispresent at a concentration of 100 microgram/ml to 0.5 mg/ml. In anotherembodiment, the present invention further provides that the bioactivecompound is present at a concentration of 0.5 mg/ml to 1 mg/ml. Inanother embodiment, the present invention further provides that thebioactive compound is present at a concentration of 0.1 mg/ml to 1mg/ml. In another embodiment, the present invention further providesthat the bioactive compound is present at a concentration of 0.5 mg/mlto 1 mg/ml.

In another embodiment, the present invention further provides that thenanoparticles and/or nanocapsules or any composition comprising thenanoparticles and/or nanocapsules is/are devoid of a low molecularweight surfactant. In another embodiment, the present invention furtherprovides that the nanoparticles and/or nanocapsules or any compositioncomprising the nanoparticles and/or nanocapsules is/are devoid of a lowmolecular weight surfactant.

In another embodiment, a composition of the invention is devoid of anorganic solvent. In another embodiment, a composition of the inventionis devoid of alcohol. In another embodiment, a composition of theinvention in the form of a solution is free of a low molecular weightemulsifier. In another embodiment, a composition of the inventioncomprises a water miscible solvent such as ethanol or DMSO in a traceamount.

In another embodiment, a composition of the invention comprises lessthan 5% in weight a water miscible solvent such as ethanol. In anotherembodiment, a composition of the invention comprises less than 2.5% in awater miscible solvent such as ethanol. In another embodiment, acomposition of the invention comprises less than 1% in weight of a watermiscible solvent such as ethanol. In another embodiment, a compositionof the invention comprises less than 0.5% in weight a water misciblesolvent such as ethanol. In another embodiment, a composition of theinvention comprises less than 0.1% in weight a water miscible solventsuch as ethanol.

In another embodiment, a composition of the invention is devoid of analcohol. In another embodiment, nanoparticles and/or nanocapsules areformed and the bioactive is entrapped simultaneously in one stage. Inanother embodiment, the process of making the nanoparticles and/ornanocapsules of the invention is devoid of heating.

In another embodiment, the present invention further provides a methodof supplementing a subject with a bioactive compound of the invention,comprising the step of administering to the subject a compositioncomprising: the aqueous liquid, a bioactive compound, and pectin orsugar-beet pectin, thereby supplementing a subject with a bioactivecompound. In another embodiment, the aqueous liquid comprises abioactive compound bound to pectin or sugar-beet pectin, therebysupplementing a subject with a bioactive compound. In anotherembodiment, an aqueous liquid is a transparent aqueous liquid comprisingnanocapsules or nanoparticles of the invention.

In another embodiment, a subject is a human. In another embodiment, asubject is an infant. In another embodiment, a subject is a toddler. Inanother embodiment, a subject is a pet. In another embodiment, a subjectis a farm animal. In another embodiment, a subject is a rodent.

In another embodiment, the present invention further provides a methodof supplementing a subject with a bioactive compound, such as anutraceutical or a drug, comprising the step of administering to thesubject a composition comprising: an aqueous liquid, a bioactivecompound, and pectin or sugar-beet pectin, thereby supplementing asubject with a bioactive compound. In another embodiment, thecomposition comprising a bioactive is administered orally. In anotherembodiment, the bioactive is any non-toxic food component which hasdemonstrated health benefits. In another embodiment, the bioactive isany sparingly water soluble, non-toxic food component, which hasdemonstrated health benefits.

In another embodiment, the bioactive nutraceutical is an omega-3 fattyacid such as α-linolenic acid (ALA) and/or eicosapentaenoic acid (EPA).In another embodiment, the nutraceutical is sea food PUFA such aseicosapentaenoic acid (EPA) and/or docosahexaenoic acid (DHA). Inanother embodiment, the nutraceutical is DHASCO (DHA single cell oil).In another embodiment, the nutraceutical is a monounsaturated fatty acid(MUFAs) such as oleic acid. In another embodiment, the nutraceutical ismedium-chain fatty acids (MCFAs) and/or medium-chain triacylglycerol(MCT). In another embodiment, the nutraceutical is conjugated linoleicacid (CLA) and/or γ-linolenic acid. In another embodiment, thenutraceutical is diacylglycerol (DAG) oil. In another embodiment, thenutraceutical is a triacyl glycerol (TAG). In another embodiment, thenutraceutical is a phospholipid.

In another embodiment, the present invention further provides a methodof supplementing a subject with a bioactive compound of the invention,comprising the step of administering to the subject a nano-emulsioncomposition comprising: an aqueous liquid, a bioactive compound, andpectin or sugar-beet pectin, thereby supplementing a subject with abioactive compound.

In another embodiment, the subject is afflicted with a disease requiringessential fatty acids support. In another embodiment, the subject isafflicted with a cardiovascular disease. In another embodiment, thesubject is afflicted with a reproductive disease. In another embodiment,the subject is afflicted with an immune disease. In another embodiment,the subject is afflicted with a nervous system disease.

In another embodiment, the subject is an infant and the composition isused for supplementing required essential fatty acids for neuraldevelopment and maturation of sensory systems. In another embodiment,the composition is used for supplementing required essential fattyacids/lipids for treating health conditions such as but not limited to:skin diseases and pathologies, hair loss, behavioral changes, failure toheal wounds, miscarriages, arthritic conditions, increased cholesterol,growth retardation, depression, dyslexia, impaired vision, learningproblems in children, heart attacks, cancer, insulin resistance, asthma,lupus, schizophrenia, accelerated aging, stroke, obesity, diabetes,ADHD, and alzheimer's disease, etc.

In another embodiment, the bioactive compound is an eicosanoid, anarachidonic acid, or any derivative thereof. In another embodiment, abioactive compound of the invention such as prostaglandin E 2 (PGE 2) isused to suppress the immune response of a subject. In anotherembodiment, a bioactive compound of the invention such as PGE2 is usedto promote cell growth of a subject. In another embodiment, a bioactivecompound of the invention such as PGE2 is used as a vasodilator. Inanother embodiment, a bioactive compound of the invention such as PGE2is used to induce and/or enhance the formation of anti-inflammatorylipoxins in a subject.

In another embodiment, a bioactive compound of the invention such asProstaglandin I(2) (PGI(2)) is used to suppress the immune response of asubject. In another embodiment, a bioactive compound of the inventionsuch as PGI(2) is used to inhibit platelet aggregation in a subject. Inanother embodiment, a bioactive compound of the invention such as PGI(2)is used as a potent vasodilator.

In another embodiment, a bioactive compound of the invention such asThromboxane A2 (TXA2) is used to suppress the immune response of asubject. In another embodiment, a bioactive compound of the inventionsuch as TXA2 is used as a vasoconstrictor.

In another embodiment, a bioactive compound of the invention such asProstaglandin D2 (PGD2) is used to inhibit platelet aggregation in asubject. In another embodiment, a bioactive compound of the inventionsuch as PGD2 is used as a sleep promoter in a subject. In anotherembodiment, a bioactive compound of the invention such as PGD2 is usedas a vasodilator.

In another embodiment, a bioactive compound of the invention such as12-hydroxy-5,8,10,14-eicosatetraenoic acid (12-HETE) is used as aneutrophil chemo-attractant. In another embodiment, a bioactive compoundof the invention such as 12-HETE is used as a stimulator ofglucose-induced insulin secretion. In another embodiment, a bioactivecompound of the invention such as 15-Hydroxyeicosatetraenoic acid(15-HETE) is used as an inhibitor of 5- and 12-lipoxygenase. In anotherembodiment, a bioactive compound of the invention such as Lipoxin A isused as a chemo-attractant. In another embodiment, a bioactive compoundof the invention such as Lipoxin B is used as an inhibitor of NK cellactivity.

In another embodiment, a bioactive compound of the invention such as afatty acid is used, for example, in the treatment of chronic diseasessuch as but not limited to: CHD, obesity, diabetes, and specific typesof cancers as are known to one of average skill in the art. In anotherembodiment, a bioactive compound of the invention is used, for example,in the treatment of vitamin D deficiency.

In another embodiment, the invention further provides a kit comprisingthe nanoparticles or nanocapsules of the invention in liquid or dry formand dosing, mixing, and/or formulating instructions. In anotherembodiment, the invention further provides a kit comprising thenanoparticles or nanocapsules of the invention and dosing, mixing,and/or formulating instructions with an aqueous solution as describedherein. In another embodiment, the invention further provides a kitcomprising the nanoparticles or nanocapsules, an aqueous solution asdescribed herein and dosing, mixing, and/or formulating instructions.

In one embodiment, compositions of the present invention are presentedin a pack or dispenser device, such as an FDA approved kit, whichcontain one or more unit dosage forms containing the nanoparticles ornanocapsules. In one embodiment, the pack, for example, comprise metalor plastic foil, such as a blister pack. In one embodiment, the pack ordispenser device is accompanied by instructions for administration. Inone embodiment, the pack or dispenser is accommodated by a noticeassociated with the container in a form prescribed by a governmentalagency regulating the manufacture, use or sale of pharmaceuticals and/ornutraceuticals, which notice is reflective of approval by the agency ofthe form of the compositions or human or veterinary administration. Suchnotice, in one embodiment, is labeling approved by the U.S. Food andDrug Administration for prescription drugs or of an approved productinsert.

In one embodiment, toxicity and therapeutic efficacy of thenanoparticles or nanocapsules described herein can be determined bystandard pharmaceutical procedures in vitro, in cell cultures orexperimental animals. In one embodiment, the data obtained from these invitro and cell culture assays and animal studies can be used informulating a range of dosage for use in human. In one embodiment, thedosages vary depending upon the dosage form employed and the route ofadministration utilized. In one embodiment, the exact formulation, routeof administration and dosage can be chosen by the individual physicianin view of the patient's condition. [See e.g., Fingl, et al., (1975)“The Pharmacological Basis of Therapeutics”, Ch. 1 p. 1].

In one embodiment, depending on the severity and responsiveness of thecondition to be treated, dosing can be of a single or a plurality ofadministrations, with course of treatment lasting from several days toseveral weeks or until cure is effected or diminution of the diseasestate is achieved.

In one embodiment, the amount of a composition to be administered will,of course, be dependent on the subject being treated, the severity ofthe affliction, the manner of administration, the judgment of theprescribing physician, etc.

In one embodiment, the nanocapsules and/or nanoparticles of the presentinvention can be provided to the individual per se (as a powder forexample). In one embodiment, the nanocapsules and/or nanoparticles ofthe present invention can be provided to the individual as part of apharmaceutical composition where it is mixed with a pharmaceuticallyacceptable carrier.

In one embodiment, a “pharmaceutical composition” refers to apreparation of one or more nanocapsules and/or nanoparticles describedherein with other chemical components such as physiologically suitablecarriers and excipients. The purpose of a pharmaceutical composition isto facilitate administration of nanocapsules and/or nanoparticles to anorganism.

In one embodiment, the phrases “physiologically acceptable carrier” and“pharmaceutically acceptable carrier”, which are interchangeably used,refer to a carrier or a diluent that does not cause significantirritation to an organism and does not abrogate the biological activityand properties of the administered compound. An adjuvant is includedunder these phrases. In one embodiment, one of the ingredients includedin the pharmaceutically acceptable carrier can be for examplepolyethylene glycol (PEG), a biocompatible polymer with a wide range ofsolubility in both organic and aqueous media (Mutter et al. (1979).

In one embodiment, “excipient” refers to an inert substance added to apharmaceutical composition to further facilitate administration ofnanocapsules and/or nanoparticles. In one embodiment, excipients includecalcium carbonate, calcium phosphate, various sugars and types ofstarch, cellulose derivatives, gelatin, vegetable oils and polyethyleneglycols.

Techniques for formulation and administration of drugs are found in“Remington's Pharmaceutical Sciences,” Mack Publishing Co., Easton, Pa.,latest edition, which is incorporated herein by reference.

In one embodiment, suitable routes of administration, for example,include oral, rectal, transmucosal, transnasal, intestinal or parenteraldelivery, including intramuscular, subcutaneous and intramedullaryinjections as well as intrathecal, direct intraventricular, intravenous,intraperitoneal, intranasal, or intraocular injections.

Oral administration, in one embodiment, comprises a unit dosage formcomprising solutions, suspensions, emulsions and the like. Such unitdosage forms comprise a safe and effective amount of the desirednanocapsules and/or nanoparticles.

Peroral compositions, in some embodiments, comprise liquid solutions,emulsions, suspensions, and the like.

In some embodiments, compositions for use in the methods of thisinvention comprise solutions or emulsions, which in some embodiments areaqueous solutions or emulsions comprising a safe and effective amount ofthe nanocapsules and/or nanoparticles of the present invention andoptionally, other compounds. In some embodiments, the compositionscomprise from about 0.01% to about 10.0% w/v of a subject compound, morepreferably from about 0.1% to about 2.0.

Further, in another embodiment, the pharmaceutical compositions areadministered topically to body surfaces, and are thus formulated in aform suitable for topical administration. Suitable topical formulationsinclude gels, ointments, creams, lotions, drops and the like. Fortopical administration, the nanocapsules and/or nanoparticles of thepresent invention are combined with an additional appropriatetherapeutic agent or agents, prepared and applied as solutions,suspensions, or emulsions in a physiologically acceptable diluent withor without a pharmaceutical carrier.

In one embodiment, pharmaceutical compositions of the present inventionare manufactured by processes well known in the art, e.g., by means ofconventional mixing, dissolving, granulating, dragee-making, levigating,emulsifying, encapsulating, entrapping or lyophilizing processes.

In one embodiment, injectables, of the invention are formulated inaqueous solutions. In one embodiment, injectables, of the invention areformulated in physiologically compatible buffers such as Hank'ssolution, Ringer's solution, or physiological salt buffer. In someembodiments, for transmucosal administration, penetrants appropriate tothe barrier to be permeated are used in the formulation. Such penetrantsare generally known in the art.

In one embodiment, the preparations described herein are formulated forparenteral administration, e.g., by bolus injection or continuousinfusion. In some embodiments, formulations for injection are presentedin unit dosage form, e.g., in ampoules or in multidose containers withoptionally, an added preservative. In some embodiments, compositions aresuspensions, solutions or emulsions in aqueous vehicles, and containformulatory agents such as suspending, stabilizing and/or dispersingagents.

The compositions also comprise, in some embodiments, preservatives, suchas benzalkonium chloride and thimerosal and the like; chelating agents,such as edetate sodium and others; buffers such as phosphate, citrateand acetate; tonicity agents such as sodium chloride, potassiumchloride, glycerin, mannitol and others; antioxidants such as ascorbicacid, acetylcystine, sodium metabisulfote and others; aromatic agents;viscosity adjustors, such as polymers, including cellulose andderivatives thereof; and polyvinyl alcohol and acid and bases to adjustthe pH of these aqueous compositions as needed. The compositions alsocomprise, in some embodiments, local anesthetics or other actives. Thecompositions can be used as sprays, mists, drops, and the like.

In another embodiment, the pharmaceutical composition is delivered in acontrolled release system formulated for intravenous infusion,implantable osmotic pump, transdermal patch, or other modes ofadministration. In one embodiment, a pump is used (see Langer, supra;Sefton, CRC Crit. Ref. Biomed. Eng. 14:201 (1987); Buchwald et al.,Surgery 88:507 (1980); Saudek et al., N. Engl. J. Med. 321:574 (1989).Other controlled release systems are discussed in the review by Langer(Science 249:1527-1533 (1990).

In some embodiments, the nanocapsules and/or nanoparticles are in powderform and possibly in kits for constitution with a suitable vehicle,e.g., sterile, pyrogen-free water based solution or a beverage, beforeuse. Compositions are formulated, in some embodiments, for atomizationand inhalation administration. In another embodiment, compositions arecontained in a container with attached atomizing means.

In one embodiment, the preparation of the present invention isformulated in rectal compositions such as suppositories or retentionenemas, using, e.g., conventional suppository bases such as cocoa butteror other glycerides.

In one embodiment, nanocapsules or nanoparticles of the invention aremade by a process comprising the steps of: preparing solution 1comprising: (a) dissolving pectin or sugar-beet pectin in water at aconcentration of 0.1-100 g/L and typically at a concentration of 0.7 to1.5 g/L; (b) stirring/mixing the solution for 20 minutes to 20 hours andtypically for 0.5 to 2 hours at 4 to 40° C. and typically at 25° C.; (c)after complete dissolution the solution is filtered through a filterhaving a cutoff of 0.1 to 2 micron; preparing solution 2 comprising: (a)dissolving the bioactive compound in a water-miscible organic food gradesolvent (typically absolute ethanol), or a water-miscible solvent forpharmaceutical applications (e.g. DMSO); combining solution 1 andsolution 2 by drop-wise or slowly adding solution 2 into solution 1 andvigorous stirring. In another embodiment, vigorous stirring is performedby utilizing vortex.

In another embodiment, the final concentration of the organic solvent inthe obtained aqueous solution is 0.001 to 20% and more typically 0.1 to2%. In another embodiment, the final concentration of the organicsolvent in the obtained aqueous solution is 0.01 to 10%. In anotherembodiment, the final concentration of the organic solvent in theobtained aqueous solution is 0.01 to 1%. In another embodiment, thefinal concentration of the organic solvent in the obtained aqueoussolution is 0.1 to 5%. In another embodiment, the final concentration ofthe organic solvent in the obtained aqueous solution is 0.1 to 5%. Inanother embodiment, the final concentration of the organic solvent inthe obtained aqueous solution is 0.1 to 1%. In some embodiments, theorganic solvent is completely removed by evaporation and/or drying.

In another embodiment, the combined solution 1 and solution 2 is furtherfiltered through a filter having cutoff of 0.1 to 15 micron. In anotherembodiment, the filter of the invention has a cutoff of 0.2 to 5 micron.In another embodiment, the filter of the invention has a cutoff of 0.1to 0.8 micron. In another embodiment, the filter of the invention has acutoff of 0.2 to 0.5 micron. In another embodiment, the filter of theinvention has a cutoff of 0.1 to 0.45 micron.

In another embodiment, the combined mix of solution 1 and solution 2,filtered or unfiltered, is further dried according to methods known inthe art and a powder is obtained. In another embodiment, the combinedmix of solution 1 and solution 2, filtered or unfiltered, is freezedried. In another embodiment, a cryoprotectant (e.g. trehalose ormaltodextrin) is further utilized. In another embodiment, the combinedmix of solution 1 and solution 2, filtered or unfiltered, is quenchfrozen (e.g. by liquid nitrogen). In another embodiment, a powdercomprising or consisting the resulting nanocapsules or nanoparticles ofthe invention is obtained. In another embodiment, a powder comprising orconsisting the resulting nanocapsules or nanoparticles of the inventionis reconstituted by adding a known amount of the powder to an aqueoussolution, while stirring, thereby obtaining a composition of theinvention.

EXAMPLES Example 1 Sugar Beet Pectin-Based Delivery Systems for FoodEnrichment with Nutraceuticals to Improve Human Health

Enhancing health-promoting properties of food may be attainable byincorporating nutraceutical delivery systems. To this end,nanoencapsulation offers several advantages, including stabilization andprotection of hydrophobic nutraceuticals even in transparent beverages,as well as their controlled and targeted release in the humangastrointestinal tract. Sugar beet pectin, a by-product of sugarproduction from sugar beet, is a readily sourced, naturally occurringdietary fiber with high emulsifying capacity. The exact underlyingmolecular traits delineating sugar beet pectin functionality are stillobscure.

Sugar Beet Pectin (SBP) Critical Micellization Concentration (CMC) andCurcumin Binding Constant

The self-assembly of SBP was studied using pyrene—a hydrophobic domainprobe (Aguiar, J.; Carpena, P.; Molina-Bolivar, J. A.; Ruiz, C. C. Onthe determination of the critical micelle concentration by the pyrene1:3 ratio method Journal of Colloid and Interface Science 2003, 258,(1), 116-122.). The ratio between the emission intensity of the third(˜383 nm) and first (˜373 nm) peaks (I3/I1) (excitation at 338 nm) weremeasured at rising SBP concentration (C). The critical micellizationconcentration (CMC) was estimated from the onset of rise in the I3/I1vsSBP concentration, to be ˜1 mg/ml. The binding constant obtained(FIG. 1) using a Langmuir model was K_(b)=(6.74±0.5)*10⁵ M⁻¹, showinghigh affinity, and suggesting that SBP is an excellent entrapping agentfor delivery of hydrophobic bioactives, e.g. curcumin (FIG. 2).

SBP Protects Curcumin During Simulated Product Shelf Life

As can be seen in FIG. 3, unprotected curcumin decreased byapproximately 40% over a period of 11 days, while curcumin encapsulatedin SBP showed only a 15% decrease. These results indicate a remarkableprotection of curcumin by SBP, suggesting that SBP may be a usefulprotective nanoencapsulation agent for enriching food & beverageproducts with curcumin, and possibly similar hydrophobic nutraceuticals.

CONCLUSIONS

SBP showed a CMC of about 1 mg/ml. Binding of curcumin to SBP was foundto be of high affinity (k_(b)=(6.74±0.5)·10⁵M⁻¹), suggesting that SBPhas very good binding properties, useful for effective delivery ofimportant hydrophobic bioactives.

Moreover, while unprotected curcumin decreased by approximately 40% overa period of 11 days, curcumin encapsulated in SBP showed only a 15%decrease. These results indicate a remarkable protection of curcumin bySBP, suggesting that SBP may be a useful protective nanoencapsulationagent for enriching food & beverage products with curcumin, and possiblysimilar hydrophobic nutraceuticals.

What is claimed is:
 1. A method of supplementing a subject with a bioactive compound, comprising the step of administering to said subject a composition comprising: a transparent aqueous liquid and a nanoparticle, said nanoparticle comprises: (a) a bioactive compound, and (b) sugar-beet pectin, wherein said bioactive compound is bound to said sugar-beet pectin, thereby supplementing a subject with a bioactive compound.
 2. The method of claim 1, wherein said composition is in the form of a nano-emulsion or a nano-dispersion.
 3. The method of claim 1, wherein said subject is afflicted with an essential fatty acid deficiency or a vitamin D deficiency.
 4. The method of claim 1, wherein said bioactive compound is a compound having a maximal aqueous solubility below 1 g/l in its free form.
 5. The method of claim 1, wherein said bioactive compound is an oil-soluble vitamin, a polyunsaturated fatty acid or its ester, an antioxidant, or a phytochemical.
 6. The method of claim 1, wherein said bioactive compound is an essential fatty acid or its ester, an Omega-3 fatty acid or its ester, docosahexaenoic acid (DHA) or its ester, eicosapentaenoic acid (EPA) or its ester, or any combination thereof.
 7. The method of claim 1, wherein said bioactive compound is: vitamin D, vitamin E, vitamin A, vitamin K, curcumin; coenzyme Q-10, a carotenoid, an isoflavone or polyphenol, or any combination thereof.
 8. The method of claim 1, wherein said nanoparticle is of a a diameter of 10 to 100 nm.
 9. The method of claim 1, wherein said sugar beet pectin is an unfractioned sugar beet pectin.
 10. The method of claim 1, wherein said composition is a beverage.
 11. The method of claim 1, wherein said composition is devoid of an additional emulsifier.
 12. The method of claim 1, wherein said bioactive compound is present at a concentration of 0.01 microgram/ml to 10 mg/ml. 